Method for preparing radiolabeled thymidine having low chromophoric byproducts

ABSTRACT

The invention is a method and related precursor for preparing  18 F-FLT. The precursor has a butoxycarbonyl protecting group at the 5′-position that results in low amounts of chromophoric byproducts being formed during deprotection. The method for preparing  18 F-FLT is efficient and makes the final purification step less complicated.

BACKGROUND OF THE INVENTION

Positron emission tomography (PET) is a diagnostic imaging technique formeasuring the metabolic activity of cells in the human body. PET canshow images of blood flow, glucose metabolism in the brain, or rapidchanges in activity in various areas of the body. It can be used to showchanges in physiology before any change in gross anatomy has occurred.PET has been used in diagnosing diseases such as cancer, heart disease,Alzheimer's disease, Parkinson's disease, and schizophrenia.

PET uses chemical compounds that are labeled with radioactive atoms thatdecay by emitting positrons. The most commonly used PET radioisotopesare ¹¹C, ¹³N, ¹⁵O, and ¹⁸F. Typically, the labeled compound is a naturalsubstrate, substrate analog, or drug that is labeled with a radioisotopewithout altering the compound's chemical or biological properties. Afterinjection into the tissue, the radiolabeled compound should follow thenormal metabolic pathway of its unlabeled counterpart. The labeledcompound emits positrons as it moves through the tissue. Collisionsbetween the positrons and electrons that are present in the tissue emitgamma rays that are detectable by a PET scanner.

Radiolabeled thymidine is a PET tracer that is useful for imagingtumors. In particular, 3′-Deoxy-3′-[¹⁸F]-fluoro-thymidine (¹⁸F-FLT) hasbeen used for visualizing DNA replication in humans and animals. ¹⁸F-FLTis incorporated into DNA during the synthesis phase of the cell cycleand therefore is a useful indicator of cellular proliferation.

After injection into a patient, ¹⁸F-FLT is taken up by cells andundergoes phosphorylation by thymidine kinase-1 (TK), an enzyme that isexpressed during cellular DNA synthesis. The phosphorylated FLT moleculeis retained within the cell, which results in its accumulation. As aresult, ¹⁸F-FLT provides insight into cellular activity and is anexcellent proliferation marker for PET tumor studies.

The usefulness of ¹⁸F-FLT as a tumor imaging agent has resulted in aneed to develop methods for its quick and efficient synthesis. Typicalmethods for preparing ¹⁸F-FLT have low reaction yields. Newer methodsthat increase yield require more synthetic transformations. Often, thesenewer methods result in chromophoric byproducts that are produced duringthe synthesis. For instance, dimethoxy trityl (DMT) is a protectinggroup that is often used to protect the 5′-hydroxy. During deprotection,DMT-OH and DMT cation are generated. DMT cation has an orange color. Theformation of DMT cation is a typical example of chromophoric byproductsthat can be produced during the deprotection step.

The presence of chromophoric byproducts complicates the purificationprocess and makes it more difficult and expensive to efficiently produce¹⁸F-FLT. During purification, the radiolabeled product is typicallyloaded onto a reverse phase column and eluted. If there is a largeamount of byproducts, the byproducts can bleed into the final productproducing an impure final product.

BRIEF SUMMARY OF THE INVENTION

The invention is a method for preparing radiolabeled nucleosides, and inparticular, ¹⁸F-FLT and related precursors. The method allows thesynthesis in a short number of steps with high yield.

The invention uses an ¹⁸F-FLT precursor that produces low amounts ofchromophoric byproducts during the deprotection step. It has beendiscovered that using a t-butoxycarbonyl protecting group at the5′-position provides several advantages. Using t-butoxycarbonyl at the5′-position results in the formation of less chromophoric byproductsduring the deprotection step. During deprotection, t-butoxycarbonyl canbe converted into t-BuOH and CO₂. Both t-BuOH and CO₂ are colorless andvolatile. As a result, the amount of byproducts that are generated isdecreased. Thus, the invention provides a method and precursor thatproduce ¹⁸F-FLT in high yield with a purification process that is lesscomplicated and more efficient.

The precursor has the following formula:

wherein P is an amine protecting group and L is a leaving group.

The preparation of ¹⁸F-FLT can begin by converting thymidine into2,3′-anhydrothymidine. The 2,3′ anhydro ring is opened to produce3′-beta hydroxyl thymidine. In the next steps, the 5′-hydroxyl group isprotected, the 3′-hydroxy group is converted into a leaving group, andthe 3-N amine is protected with an amine protecting group.

The precursor is immediately ready for radiolabeling or alternativelycan be stored for future use. After radiolabeling, the protecting groupsare removed. Typically, the removal is carried out with acid hydrolysis.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings wherein:

FIG. 1 illustrates a reaction scheme for preparing ¹⁸F-FLT; and

FIG. 2 is structural drawing of thymidine showing the numbering ofcarbon atoms in the compound.

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements.

DETAILED DESCRIPTION OF THE INVENTION

The synthesis of radiolabeled nucleosides can begin with a nucleosidehaving a pyrimidine base, such as thymidine, uridine, or cytidine. Inthe invention the nucleoside with a pyrimidine base is converted into ananhydronucleoside.

FIG. 1 illustrates an exemplary reaction scheme for preparing ¹⁸F-FLT inaccordance with the invention. For ease of discussion, FIG. 2,illustrates a thymidine molecule in which the carbon atoms have beennumbered. The numbering convention shown in FIG. 2 is used throughoutthe disclosure. It should be recognized that the carbon atoms could benumbered differently and that the invention is not limited by anyparticular numbering format.

The synthesis of ¹⁸F-FLT and its related precursor can begin withthymidine. As illustrated in FIG. 1, the method of preparing ¹⁸F-FLTcontains the following steps outlined below:

a. converting thymidine into 2,3′-anhydrothymidine;

b. opening the 2,3′-anhydro ring;

c. protecting the 5′-hydroxyl group with t-butoxycarbonyl (boc);

d. incorporating a leaving group at the 3′-position;

e. protecting the 3-N amine group;

f. radiolabeling the ¹⁸F-FLT precursor; and

g. removing the amine protecting group and boc at the 5′-position.

Depending upon reaction conditions, the order of steps c, d, and e canbe varied, although not necessarily with equivalent results. Each stepis discussed in greater detail below.

In the first step, thymidine is converted into 2,3′-anhydrothymidine toproduce a compound having the following formula:

2,3′-anhydrothymidine can be a useful starting compound because itplaces the 3′-hydroxyl group in the beta position so that a leavinggroup can also be positioned in the beta position in a subsequentreaction. As a result, when fluorinated, ¹⁸F can attack the 3′-carbonanti to the leaving group and can be attached to the 3′-carbon in the αorientation.

There are a variety of known techniques for converting a nucleoside intoan anhydronucleoside. For example, the anhydronucleoside can be preparedby mixing thymidine with triphenylphosphine and azeotropically dryingwith portions of acetonitrile (MeCN). The resulting mass is suspended inMeCN and then cooled. The mixture is rapidly stirred anddiisopropylazodicarcobxylate in MeCN is added dropwise to the mixture.The resulting mixture is treated with water to form a suspension that isfiltered to afford anhydrothymidine. (Grierson, J. R., Shields, A. F.,Nuclear Medicine and Biology, 2000, 27 143-156; Balagopala, M. I.,Ollapally, A. P., and Lee, H. J., Nucleosides-Nucleotides, 1996, 15(4)899-906).

U.S. Pat. No. 5,717,086 discloses a method of converting a nucleosideinto a 2,3′-anhydronucleoside by reacting it with a dehydrating agent inthe presence of an acid. Specifically, it discloses that 2′-deoxyuridinemay be reacted with a combination of diisopropylazodicarboxylate ordiethylazodicarboxylate and a triaryl- or trialkyl-phosphine or-phosphite, e.g., triphenylphosphine, preferably in the presence of anacid, in an inert polar solvent. It should be recognized that there aremany different methods that can be used to convert a nucleoside into itsanhydronucleoside derivative, although not necessarily with equivalentresults.

Alternatively, the synthesis may begin with a commercially available2,3′-anhydro nucleoside, such as 2,3′-anhydrothymidine or one of itsderivatives. It should be recognized that the invention can also includenucleoside derivatives that contain additional substituents providedthat the substituents are non-interfering and do not prevent, block, ornegatively impact the reactivity or functionality of the precursor, anyreaction steps, or the final product. Such derivatives are known in theart and include, without limitation, deuterated derivatives, such as2′-deuterated nucleosides, or derivatives having different substituentsat the 5-position, such as bromomethyl, benzyl, or the like.

After converting thymidine into 2,3′-anhydrothymidine, the 2,3′-anhydroring is opened to produce 3′-beta-hydroxyl thymidine. Typically, thering is opened using a basic reagent such as NaOH, KOH, LiOH,alkylammonium hydroxides such as tetrabutylammonium hydroxide, andresins such as Amberlite IRA 400 (OH—).

In the next step, the 5′-hydroxy is protected with a protecting group toproduce a compound having the following formula:

It has been discovered that protecting the 5′-hydroxyl witht-butoxycarbonyl results in the formation of fewer chromophoricbyproducts during deprotection. As a result, yield is higher and thepurification process is easier and more efficient.

In an alternative embodiment, the 5′-hydroxyl can be protected withprotecting groups other than t-butoxycarbonyl to produce the followingintermediate:

wherein X is a hydroxyl protecting group. Typically, suitable protectinggroups should not contain extensive conjugation and should decomposeinto compounds that are volatile and colorless. Useful protecting groupsinclude carbonates, such as methyl, methoxymethyl ethyl, and isobutyl;cyclic ethers, such as tetrahydropyranyl ether and tetrahydrafuranylether; and alkyl ethers, such as methoxymethyl ether,bis-(2-chloroethoxy)methyl ether, 1-ethoxyethyl ether, and1-methyl-1-methoxyethyl ether.

A leaving group is incorporated at the 3′-position to produce theprecursor having the following formula:

wherein L is a leaving group.

The leaving group activates the thymidine derivative and is replacedduring the [¹⁸F] fluorination step. During the radiolabeling step, [¹⁸F]fluoride attacks the 3′-carbon atom anti to the 3′-leaving groupresulting in substitution of the leaving group by a bimolecularnucleophile substitution mechanism (S_(N)2).

Leaving groups that are useful in the invention are moieties that can bedisplaced from the 3′-carbon atom by nucleophilic substitution. Theleaving group should attach to the 3′-hydroxyl or replace it to form aleaving group at the 3′-postion. The leaving group moiety should notreact with other sites or functional groups that may be present on thethymidine derivative. The leaving group should also be able to bequickly replaced by the radioisotope during the radiolabeling step.Typically, the leaving group should be replaced by [¹⁸F]fluoride inpolar aprotic solvent.

The term leaving group (“L”) refers to moieties that should besusceptible to displacement by a nucleophile, wherein the 3′-hydroxy canattach to another substituent directly to form a leaving group or the3′hydroxy may be removed in order to incorporate the leaving group.Sulfonate ester is an exemplary leaving group that is formed from asulfonyl moiety attaching directly to the 3′-hydroxy.

Useful leaving groups that combine with the 3′-hydroxy include, withoutlimitation, sulfonyl moieties, such as alkylsulfonyl, substitutedalkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,hetercyclosulfonyl or trichlorcacetimidate groups. Particularly usefulgroups include, without limitation, benzenesulfonyl, methylsulfonyl(mesylate), p-methylbenzenesulfonyl (tosylate), 4-nitrobenzene sulfonyl(nosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, 2,2,2-trifluoroethanesulfonyl, andimidazolesulfonyl. It should be recognized that other moieties can beused to form —O-L′ leaving groups, although not necessarily withequivalent results.

Other useful ¹⁸F-FLT precursors include thymidine derivatives whereinthe 3′hydroxy has been completely replaced with an alternative leavinggroup, such as a halogen.

The 3-N amine group is protected with an amine protecting group toproduce an ¹⁸F-FLT precursor having the following formula:

wherein P is an amine protecting group and L is the same as definedabove.

Amine protecting groups that are useful in the invention should possesslow chromophoric activity, prevent unwanted reactions towards the aminegroup, are not affected by reaction conditions, do not interfere withreactions on other portions of the molecule, and are easily removed atthe end of the reaction scheme or at any other time that is appropriate.It is also desirable that the protecting group will help enhancereactions by increasing yield or selectivity.

Useful amine protecting groups include, without limitation, carbamates,such as tert-butoxycarbamate, isopropyl carbamate, pivaloyloxymethylcarbamate, and allyl carbamate; cyclic ethers, such as N-tetrahydropyranand N-tetrahydrofuran; cyclic alkyl ethers, such as t-butylamide; andcyclic amines, such as N-pyrrolidinomethylamide. Exemplary amineprotecting groups include, without limitation, t-butoxycarbamate,pivaloyloxymethyl carbamate, allyl carbamate, and methyl carbamate.

A particularly useful protecting group is t-butoxycarbonyl (boc).Typically, the 3-N amine group is protected by reacting the thymidineprecursor with Boc₂O in N,N-dimethylformamide. Boc is a particularlyuseful amine protecting group because it reacts quickly with the aminegroup, does not interfere in the fluorination step, and is easilyremoved with hydrolysis.

The precursor is now ready for immediate [¹⁸F]fluorination or can bestored and transported for future use. The precursor is shelf stable andis highly reactive with [¹⁸F]fluoride in polar aprotic solvents.

Radiolabeling can be carried out using a variety of methods. In themethod for preparing ¹⁸F-FLT, the precursor is treated with Kryptofix222® and potassium carbonate in the presence of a polar aprotic solvent(Wodarski, C., et al., J. Labelled Cpd. Radiopharm., 2000, 43 1211-1218;Blocher A., et al., J. of Radioanalytical and Nuc. Chemistry, 2002,251(1), 55-58; Martin, S. J. et al., Nuclear Medicine and Biology, 2002,29 263-273). Useful solvents include, without limitation, acetonitrile,pyridine, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), andblends thereof.

In the final step of preparing ¹⁸F-FLT, the 3-N protecting group and5′-boc group are removed. Typically, the boc and amine protecting groupare removed by hydrolyzing the radiolabeled nucleoside. Usefulhydrolyzing reagents include, without limitation, acids, such as HCl,HBr, HOAc, H₂SO₄, HI, trimethylsilyliodide (TMSI), and H₃PO₄.

As should be evident from the above disclosure, the method would also beuseful for preparing other radiolabeled nucleoside compounds that have apyrimidine base. For example, the method could be used to prepareradiolabeled derivatives of uridine and cytidine. In preparingradiolabeled cytidine and uridine derivatives, the synthesis shouldbegin with their 2′-deoxy derivatives.

EXAMPLE Synthesis of the ¹⁸F-FLT Precursor

Step a: Synthesis of Anhydrothymidine.

To a dried round bottom flask containing thymidine(10.0 g, 41.6 mmol)was added triphenylphosphine (21.8 g, 82.2 mmol) and CH₃CN (160 mL). Thesuspension was cooled to −20° C. ((40:60 iPrOH:H₂O) and dry ice). To thereaction mixture was added diisopropylazodicarboxylate (16.8 g, 82.2mmol) as CH₃CN solution (60 mL) dropwise via an addition funnel, over aperiod of 1 hour. After the addition, the mixture was stirred for anadditional 90 minutes at 20° C. The mixture was allowed to warm to 10°C. over a period of 5 hours. The reaction was then quenched with H₂O (6mL) forming a white suspension. The reaction was allowed to stand for 30minutes and then was filtered. The collected solid was washed with coldCH₃CN and dried under vacuum to afford 7.7 g (83% yield) of a whitesolid. If desired, the solid may be recrystallized in EtOH.

Step b: Synthesis of 3′-β-hydroxy thymidine.

To a round bottom flask containing anhydrothymidine (3.39 g, 15 mmol)and water (20 mL) was added tetrabutylammonium hydroxide (TBAM) (30 mL,30 mmol). The solution was allowed to stir at room temperatureovernight. LC/MS indicated no anhydrothymidine was present. To thesolution was added enough IRA H+ resin to neutralize all the TBAM. Theresin was filtered off and washed with methanol. The filtrate wasconcentrated to dryness with toluene to azeotrope off the water. Theresidue was purified via recrystallization from acetonitrile to afford3.1 g (85%) of a white precipitant.

Step c: Synthesis of 5′-O-Boc-3′-β-hydroxy thymidine.

To a dried round bottom flask was added 3′-β-hydroxy thymidine (242 mg,1.0 mmol), DMF (5 mL), pyridine (355 μL, 5 mmol) and Boc₂O (262 mg, 1.2mmol). The reaction was stirred overnight at room temperature. Thereaction was then poured onto water and extracted into CH₂Cl₂. Theorganics were combined, dried (NgSO₄), filtered, and concentrated todryness. The residue was purified on silica gel using EtOAc as theeluent to afford 115 mg (37% yield) of a white solid. ¹H NMR (300 MHz,CDCl₃) δ: 1.49 (9H, s), 1.92(3H, s), 2.21 (1H, dd, J=15.17, 2.52 Hz),2.61-2.68 (1H, m), 3,74 (1H, d, J=3.46 Hz), 3.99-4.01 (1H, m), 4.30 (1H,dd, J=11.7, 5.4 Hz), 4.36-4.38 (1H, m), 4.55 (1H, dd, J=11.7, 7.0 Hz),6.13 (1H, d, J=8.6, 2.6 Hz), 7.67 (1H, s), 8.58 (1H, s).

Step d: Synthesis of 5′-O-Boc-3′-β-nosyl thymidine.

To a dried round bottom flask was added 5′-O-Boc-3′-β-hydroxy thymidine(500 mg, 1.46 mmol) and pyridine (7 mL). The flask was cooled to 0° C.Silver triflate (745 mg, 2.9 mmol) was added in a single portionfollowed by nosyl chloride (643 mg, 2.9 mmol). The reaction was stirredat 0° C. for 1 hour and then warmed to room temperature and stirredovernight. TLC (EtOAc) indicated incomplete consumption of the startingmaterial. A second addition of silver triflate (745 mg, 2.9 mmol) wasadded followed by nosyl chloride (643 mg, 2.9 mmol) and the reaction wasstirred for 1 hour. The mixture was poured onto water and extracted intoEtOAc. The combined organic extracts were dried (MgSO₄), filtered, andconcentrated to dryness in vacuo. The residue was purified on silica gelusing 75% Hex:EtOAc as the eluent to afford 330 mg (43%) of a whitesolid. MS: Calc'd for C₂₁H₂₅N₃O₁₁S: 527.12; found: 550 (M+Na). ¹H NMR(300 MHz, CD₃Cl) δ: 1.46 (9H, s), 1.89 (3H, s), 2.41-2.44 (1H, m),2.75-2.81 (1H, m), 4.22-4.27 (2H, m), 4.39-4.43 (1H, m), 5.22 (1H, brs), 6.18-6.21 (1H, m), 7.37 (1H, s), 8.07-8.13 (2H, m), 8.38-8.42 (2H,m), 9.31 (1H, s).

Step e: Synthesis of 5′-O-Boc-3′-β-nosyl-2-N-Boc thymidine.

To a dried round bottom flask containing 5′-O-Boc-3′-β-nosyl thymidine(120 mg, 0.23 mmol), N,N-dimethylformamide (5 mL), and Boc₂O (199 mg,0.91 mmol) was added 4-N,N-dimethylaminopyridine (DMAP) (112 mg, 0.91mmol). The reaction was stirred overnight at room temperature. TLC(EtOAc:Hex 1:1) indicated complete consumption of the starting material.The reaction was poured onto water and extracted into EtOAc. Thecombined organic extracts were dried (MgSO₄), filtered, and concentratedto dryness in vacuo. The residue was purified on silica gel usingEtOAc:Hex 25:75 as the eluent to afford 69 mg (48%) of a white solid.MS: Calc'd for C₂₆H₃₃N₃O₁₃S: 627.17; found: 650.2 (M+Na). 1H NMR (300MHz, CD3CN) δ: 1.43 (9H, s), 1.53 (9H, s), 1.93 (3H, s), 2.22 (1H, d,J=16.0 Hz), 2.72-2.76 (1H, m), 4.23-4.31 (4H, m), 5.32-5.33 (1H, m),7.37 (1H, s), 8.07-8.13 (2H, m), 8.36-8.38 (2H, m).

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

1-24. (canceled)
 25. A compound having the following formula:

wherein X is t-butoxycarbonyl, methyl, methoxymethyl ethyl, isobutyl,tetrahydropyranyl ether, tetrahydrafuranyl ether, methoxymethyl ether,bis-(2-chloroethoxy)methyl ether, 1-ethoxyethyl ether, or1-methyl-1-methoxyethyl ether; P is an amine protecting group; and L isa leaving group.
 26. A compound according to claim 25, wherein P isselected from the group consisting of tert-butoxycarbamate, isopropylcarbamate, pivaloyloxymethyl carbamate, methyl carbamate, allylcarbamate, N-tetrahydropyran, N-tetrahydrofuran; t-butylamide, andN-pyrrolidinomethylamide.
 27. A compound according to claim 25, whereinP is t-butoxycarbonyl.
 28. A compound according to claim 25, wherein Lis a sulfonate ester.
 29. A compound according to claim 25, wherein L isselected from the group consisting of benzenesulfonyl, methylsulfonyl,p-methylbenzenesulfonyl, 4-nitrobenzene sulfonyl,p-bromobenzenesulfonyl, trifluoromethylsulfonyl, trichloroacetimidate,2,2,2-trifluoroethanesulfonyl, and imidazolesulfonyl.
 30. A compoundaccording to claim 25, wherein L is nosylate, tosylate, or mesylate 31.A compound according to claim 25, wherein X is t-butoxycarbonyl, methyl,methoxymethyl ethyl, or isobutyl.
 32. A compound having the followingformula:

wherein P is an amine protecting group and L is a leaving group.
 33. Acompound according to claim 32, wherein P is selected from the groupconsisting of tert-butoxycarbamate, isopropyl carbamate,pivaloyloxymethyl carbamate, methyl carbamate, allyl carbamate,N-tetrahydropyran, N-tetrahydrofuran, t-butylamide, andN-pyrrolidinomethylamide.
 34. A compound according to claim 32, whereinP is t-butoxycarbonyl.
 35. A compound according to claim 32, wherein Lis a sulfonate ester.
 36. A compound according to claim 32, wherein L isselected from the group consisting of benzenesulfonyl, methylsulfonyl,p-methylbenzenesulfonyl, 4-nitrobenzene sulfonyl,p-bromobenzenesulfonyl, trifluoromethylsulfonyl, trichloroacetimidate,2,2,2-trifluoroethanesulfonyl, and imidazolesulfonyl.
 37. A compoundaccording to claim 32, wherein L is nosylate, tosylate, or mesylate. 38.A compound having the following formula:

39.-50. (canceled)